Figure 4.

Dissecting PolyUb Binding of ZUFSP

(A) DUB assays of ZUFSP^cat and ZUFSP^MIU-cat with the indicated polyUbs. Reaction products were separated by SDS-PAGE and silver stained.

(B) Alignment of the MIU from ZUFSP with the consensus MIU motif (Penengo et al., 2006).

(C) Ub pull-down assay where HALO-MIU-ZHA immobilized on HaloLink resin was incubated with tetra-Ub (UB4) of different linkages. Bound material was separated by SDS-PAGE and silver stained. Asterisks denote unspecific bands.

(D) Indicated HALO-fusion proteins were incubated with UB4 and processed as in (B). Captured chain types are highlighted in the Binding Summary section.

(E) ESPRIPT sequence alignment of ZNF4 from ZUFSP with UBZ of DNA polymerase η (POLH) and FAAP20. Residues highlighted within blue frames are important for ZUFSP ZNF4 polyUb binding.

(F) Recombinant HALO-ZNF4 wild-type (WT) and various point mutants were analyzed for binding to tetra-K48 linked chains. Bound material was separated by SDS-PAGE and stained by Coomassie blue.

See also Figure S4.